Specific Interaction of DARPin with HIV-1 CANTD Disturbs the Distribution of Gag, RNA Packaging, and Tetraspanin Remodelling in the Membrane

A designed repeat scaffold protein (AnkGAG1D4) recognizing the human immunodeficiency virus-1 (HIV-1) capsid (CA) was formerly established with antiviral assembly. Here, we investigated the molecular mechanism of AnkGAG1D4 function during the late stages of the HIV-1 replication cycle. By applying stimulated emission-depletion (STED) microscopy, Gag polymerisation was interrupted at the plasma membrane. Disturbance of Gag polymerisation triggered Gag accumulation inside producer cells and trapping of the CD81 tetraspanin on the plasma membrane. Moreover, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) experiments were performed to validate the packaging efficiency of RNAs. Our results advocated that AnkGAG1D4 interfered with the Gag precursor protein from selecting HIV-1 and cellular RNAs for encapsidation into viral particles. These findings convey additional information on the antiviral activity of AnkGAG1D4 at late stages of the HIV-1 life cycle, which is potential for an alternative anti-HIV molecule.


Introduction
The human immunodeficiency virus-1 (HIV-1) group-specific antigen (Gag) polyprotein is the precursor of capsid (CA) protein (the target of Ank GAG 1D4) and is encoded by genomic RNA (gRNA; also known as full-length (FL) RNA). Gag plays an important role in viral assembly and RNA recruitment for HIV-1. Gag contains four major domains: matrix (MA), CA, nucleocapsid (NC), and p6, in addition to two spacer peptides, SP1 and SP2. Gag is myristoylated at the N-terminus of MA and contains a highly basic region (HBR) involved in targeting Gag to phosphatidylinositol 4,5-bisphosphate, PI (4,5) P2 and anchoring it to the inner leaflet of the host cell plasma membrane (PM), where viral assembly takes place [1,2].
The Gag precursor promotes HIV-1 FL RNA dimerisation in the cytoplasm and specifically targets the dimeric FL RNA to virus-assembly sites at the PM [3,4]. The virus-assembly site is composed of thousands of Gag polyprotein molecules, hundreds of Gag-polymerase (Pol) precursor proteins, 8-10 envelope (Env) protein trimers, and dimeric FL RNA [5,6]. the in silico study supporting anti-HIV-1 activity of Ank GAG 1D4, the molecular mechanisms whereby Ank GAG 1D4 inhibits HIV-1 replication still remain undeciphered. In this study, we investigated the impact of Ank GAG 1D4 on the late stages of viral replication: Gag polymerisation, RNA packaging, and CD81-based tetraspanin membrane remodelling. Our data indicated that Ank GAG 1D4 disturbed Gag distribution in PM, resulting in reduced HIV production.
A lentiviral vector CGW, a third-generation lentiviral vector, used as the backbone vector to transfer the genes for molecular scaffolds Myr (+) Ank GAG 1D4 or Myr (+) Ank A3 2D3 into target cells, was constructed and described previously [39]. Tagging of enhanced-green fluorescent protein (EGFP) on C-terminus of ankyrin supported the detection of ankyrin expression inside the cells.
The pFC14K HaloTag ® CMV Flexi vector (Promega, Madison, WI, USA) was used to produce a truncated, HIV-1 MA and CA (MACA) HaloTag ® fusion protein. MACA was amplified from pNL4-3, obtained from the NIH AIDS Research and Reference Reagent Program (Division of AIDS, NIAID, NIH) and then cloned into the pFC14K HaloTag ® vector, linearised with SgfI and EcoRI.

Production of VSV-G-Pseudotyped Lentivirus
VSV-G-pseudotyped lentiviral vector particles in this study were produced in HEK293T cells, using the calcium phosphate co-transfection method. To produce VSV-G-pseudotyped ankyrin-EGFP viral particles, HEK293T cells were seeded on 10-cm dishes (3.5 × 10 6 cells per dish), and co-transfected with the CGW transfer vector (10 µg/dish), the packaging construct pMDLg/pRRE (6.5 µg/dish), pRSV-Rev (2.5 µg/dish), and pMD.2G (3.5 µg/dish). Culture supernatant containing lentiviral vector particles were harvested at 24 and 48 h post-transfection, and concentrated by ultracentrifugation. The viral titers were determined by re-transduction of HEK293T cells with serial dilution of lentiviral vectors, and expressed as the percentage of EGFP positive cells determined by flow cytometry.

Establishment of HeLa Cells Stably Expressing Ankyrin
To generate HeLa cells stably expressing ankyrin, HeLa cells were transduced with the VSV-G-pseudotyped lentiviral vectors at MOI of 1 in a growth medium containing 8 µg/mL of Polybrene (Sigma Aldrich, Saint Louis, MO, USA). Each VSV-G-pseudotyped lentiviral vectors included VSVG-CGW-Myr (+) Ank GAG 1D4-EGFP, VSVG-CGW-Myr (+) Ank A3 2D3-EGFP. The transduced cells were then transferred to a humidified incubator, and maintained at 37 • C and 5% CO 2 for 24 h. The cells were washed three times with a fresh growth medium and further cultured in fresh growth medium. The efficiency and stability of transduction were determined by fluorescence microscope and flow cytometer.
Virion purification: Virions were purified from collected culture supernatants by ultracentrifugation through a 20% sucrose cushion at 100,000× g for 1.30 h at 4 • C. The pellets were resuspended in 160 µL of DMEM. Next, 25 µL of each virion sample was used for protein analysis, 125 µL was used for RNA extraction, and 10 µL was used for p24 quantification using a modified Genscreen TM ULTRA HIV Ag-Ab Assay (Bio-Rad, Hercules, CA, USA).
RNA extraction: RNA from concentrated virus samples was extracted using the QIAamp Viral RNA Mini Kit in the presence of 5.6 µg of carrier RNA (Poly A) according to manufacturer's recommended procedure (Qiagen, Hilden, Germany). The viral RNAs were then treated with DNase I (Thermo Scientific, Waltham, MA, USA) to eliminate the remaining DNA. Then, dried pellets containing 3 × 10 6 cells were used for total cellular RNA extraction with a High Pure RNA Isolation Kit (Roche, Basel, Switzerland) in the presence of DNase, according to manufacturer's recommended procedure.
Protein analysis: The remaining dried cell pellets were lysed to extract cellular protein using RIPA Lysis and Extraction Buffer (Thermo Scientific, Waltham, MA, USA), according to the manufacturer's instructions. Total protein was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Proteins in 100 µg of cell lysate or 25 µL of concentrated virion were separated using SDS-PAGE and transferred to nitro-cellulose membrane. HIV-1 Gag was detected with a mouse anti-CA antibody (hybridoma H183, NIH AIDS Reagent Program). Ankyrin protein expression was determined with a GFP Monoclonal Antibody. Horseradish peroxidase-conjugated (HRP) anti-mouse IgG (Seracare, Milford, MA, USA) was used as a secondary antibody. Cellular actin was detected using an HRP-conjugated mouse monoclonal anti-β-actin antibody (clone AC-15, Sigma-Aldrich, Saint Louis, MO, USA). ECL fluorescence was recorded using a ChemiDoc™ Touch Imaging System (Bio-Rad, Hercules, CA, USA).
Quantification of p24 in concentrated virions by performing p24 ELISA: The Genscreen TM ULTRA HIV Ag-Ab Assay Kit (Bio-Rad, Hercules, CA, USA) was modified by replacing conjugate 1 with a biotinylated anti-capsid (CA) antibody and pre-incubating the sample with 10% Triton X-100 (virion lysis buffer, (Thermo Scientific, Waltham, MA, USA) at 37 • C for 30 min before beginning the manufacturer's protocol.
RNA quantification: Reverse transcription (RT) was performed using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Basel, Switzerland) with 1 µg of total cellular RNA sample or 0.5 µg of virion RNA. An oligo (dT) primer was used as an RT primer for viral RNAs and GAPDH mRNA, whereas specific internal primers were required for RT of 7SL and U6 RNA. After the RT reactions, the RT products were diluted, and quantitative polymerase chain reaction (PCR) experiments were run on a CFX96 Real-Time System (Bio-Rad, Basel, Switzerland). The RT products were amplified with 40 cycles of PCR: 95 • C for 15 s 58 • C for 12 s, and 72 • C for 20 s. The pair of primers (Table 1) were used at the final concentration of 0.5 mM in the real-time PCR for FL HIV-1 RNA, MS HIV-1 RNA, HIV-1 env RNA, GAPDH, 7SL, and U6. Quantification was performed using a standard curve comprised of 10 2 to 10 9 copies of the pNL4-3 plasmid.

Determination of an Effect of Ank GAG 1D4 on HIV-1 Protease Activity
An effect of ankyrin on HIV-1 protease activity was determined using ELISA. The microtiter plate was coated with 5 µg/mL of recombinant H 6 MA-CA (His6-Matrix-Capsid) and incubated at 4 • C for 16 h. Then, the coated plate was washed with washing buffer 1 (0.05% Tween-20 in PBS) and blocked with 2% skim milk-PBS for 1 h. Next, plate was washed with washing buffer 1. The 5 µg/mL of recombinant Ank GAG 1D4 or Ank A3 2D3 (irrelevant ankyrin repeat protein) diluted in binding buffer (2% of BSA in 0.05% Tween 20-PBS) were added to coated well, then the reaction was placed at room temperature for 1 h. The H 6 MA-CA-binding activity of ankyrin was detected by rabbit anti-ankyrin polyclonal antibody (pre-absorbed with 5% of BSA in 0.05% Tween 20-PBS for 1 h) followed by goat anti-rabbit immunoglobulins-HRP (diluted in binding buffer). Besides, after 1 h incubation of Ankyrin to H 6 MA-CA coated plate, the HIV-1 protease activity was performed, the non-specific binding was washed with washing buffer 2 (1% Triton X100 (v/v) and 550 mM NaCl prepared in PBS, pH 7.4). The 6 mg/mL of soluble HIV-PR (diluted in binding buffer), while bacterial lysate BL21(D3) was used as a control, was added to the wells, and the plate was incubated at 37 • C for 1 h. Then, the wells were washed with washing buffer 2. The presence of the free C-terminus of MA after cleaving by HIV-PR was detected by an anti-MA (HB-8975) monoclonal antibody (specifically binds to the free C terminus of cleaved HIV-MA) followed by a goat anti-mouse immunoglobulins-HRP (diluted in binding buffer). The wells were then washed; followed by TMB substrate being added. The wells were then washed again with washing buffer 2 prior to adding SureBlue™ TMB Microwell Substrate (KPL) and the optical density at 450 nm (OD 450 nm) were measured after adding 1 N HCl.

Investigation of the Influence of Ank GAG 1D4 on Organisation of CD81 during HIV-1 Assembly
HeLa cells or HeLa cells stably expressing ankyrin were transduced with VSV-Gpseudotyped HIV-1 Gag-mCherry at MOI of 2 in a growth medium containing 8 µg/mL of Polybrene (Sigma-Aldrich, MO, USA). Transduced HeLa cells were incubated in humidified 5% CO 2 incubator. The excess lentiviruses were washed after 1 day of transduction. On the next following day, cells were collected to be analysed for CD81 expression using direct immunofluorescence staining and flow cytometry.
The direct immunofluorescence staining was performed in the adhered transduced HeLa cells on coverslip. Cells were washed with PBS and fixed with 4% paraformaldehyde-PBS for 10mins at room temperature. Cells were then washed twice with PBS and blocked with 10% human AB serum-PBS. Next, cells were stained with 100 µg/mL of Pacific Blue™ anti-human CD81 (Biolegend, San Diego, CA, USA). Finally, cells were washed and mounted with ProLong™ Gold Antifade Mountant (Thermo Scientific, Waltham, MA, USA). The CD81, Gag-mCherry, and Ankyrin-EGFP were visualised under a Nikon C2 plus confocal microscope. Excitation wavelengths were 405 nm for Pacific Blue, 488 nm for EGFP, and 560 nm for mCherry.
The transduced HeLa cells were also performed flow cytometry analysis to determine the surface CD81 expression by staining with 100 µg/mL of Pacific Blue™ anti-human CD81 (Biolegend, San Diego, CA, USA).

Statistical Analysis
Results are presented as the mean ± standard deviation (SD) of three independent assays. Statistical comparisons were determined by one-way analysis of variance (ANOVA) with GraphPad Prism software. Statistically significance was indicated as asterisk (*** p < 0.001; ** p < 0.01; * p < 0.1; ns, not significant).

Ank GAG 1D4 Affected Gag Production in HIV-1-Producer Cells
HEK293T cells were co-transfected with pNL4-3∆env plasmid with or without pCep4-Myr (+) Ank GAG 1D4-GFP or pCep4-Myr (+) Ank A3 2D3-GFP (1:1 molar ratio with pNL4-3∆env). Proteins were extracted from cell pellets and concentrated virus samples, and analysed by western blotting with antibodies against CA, GFP, and β-actin. The results show that both Ank GAG 1D4 and Ank A3 2D3 (non-binder ankyrin control) were expressed in transfected cells as detected based on GFP expression but were not incorporated into virions ( Figure 1A). The presence of both Ank GAG 1D4 and Ank A3 2D3 did not interfere with the Gag-processing pattern as Pr55Gag was processed to the p41 and p24 CA proteins. The accumulation of Pr55Gag and the p41 CA precursor was observed inside HEK293T cells transfected with the pNL4-3∆env and pCep4-Myr (+) Ank GAG 1D4-GFP ( Figure 1B). In contrast, in the presence of (Myr (+)) Ank GAG 1D4, little or no virion release was observed, as shown by the strong decrease of p24 intensity in viral lysate ( Figure 1A, B). Additionally, HIV-1 Gag processing efficiency from viral lysate was determined using image lab software ( Figure 1C). The gag processing efficiency in the presence of (Myr+) Ank GAG 1D4 was lower than the control cells. This evidence associates with the reduction in virion release. These results indicated that the intracellular expression of Ank GAG 1D4 impaired virion release; thus, Gag accumulated inside the cells without affecting the Gag processing. The percentage of HIV-1 Gag processing efficiency was calculated from the band intensity of HIV-1 Pr55, p41, and p24 in viral lysate using the p24/(pr55 + p41 + p24) formula. HEK293T cells transfected with pNL4-3 ∆env were used to normalise the data. Data represent the mean ± SD from the triplicate independent assay. *** p < 0.001; ns, not significant using one-way ANOVA.

Ank GAG 1D4 Is Not Associated with HIV-1 Protease Cleavage Site
According to Figure 1A, a few amounts of virion release and accumulation of Pr55Gag and the p41CA precursor inside the Gag-producing cells were observed, in the presence of Ank GAG 1D4 as shown in Figure 1A. To confirm whether the binding of Ank GAG 1D4 with HIV-1 capsid does not reside in the protease cleavage sites, Gag polyprotein processing by HIV-1 protease was determined using enzyme-linked immunosorbent assay (ELISA). In this experiment, the H 6 MA-CA recombinant protein, which contains the HIV-1 protease cleavage site between MA and CA, was used as a model. First, the specific binding of Ank GAG 1D4 with coated H 6 MA-CA was determined. The result demonstrated the specific interaction of Ank GAG 1D4 with coated H 6 MA-CA compared to Ank A3 2D3 (Figure 2A).
Next, in order to exhibit that Ank GAG 1D4 did not interfere with the HIV-1 protease cleavage site, Ank GAG 1D4 was prior incubated with coated H 6 MA-CA for 1 h to ensure that the binding of Ank GAG 1D4 with its target was achieved. Then, the HIV-1 protease was added to the bound H 6 MA-CA. The protease activity was measured by the presence of a free C-terminus of HIV-MA after cleaving with HIV-1 protease. The result confirmed that the binding of Ank GAG 1D4 with H 6 MA-CA did not interfere with the HIV-1 protease cleavage site as detected by free C-terminus of HIV-MA after cleaving with HIV-1 protease at the same levels as Ank A3 2D3 and control ( Figure 2B).

Ank GAG 1D4 Impaired Gag Distribution at the PM
As viral release was impaired by Ank GAG 1D4, we were interested in determining which steps of virus assembly were affected. In this experiment, HIV-1 Gag distribution in ankyrin-expressing HeLa cells was demonstrated by stimulated emission depletion (STED) microscopy. HeLa cells were transfected with the pFC14K-MACA-HaloTag ® plasmid with or without pCEP4-Myr (+) Ankyrin-GFP. After transfection, MACA-HaloTag ® expression was traced by staining with the HaloTag ® TMR Direct™ ligand. As expected, the MACA-HaloTag ® localised mainly at the plasma membrane ( Figure 3A). When co-expressing MACA-HaloTag ® and Myr + Ank A3 2D3 (non-binder ankyrin control) little colocalisation was observed ( Figure 3B), whereas MACA-HaloTag ® colocalised with Myr (+) Ank GAG 1D4 throughout the PM ( Figure 3C). Interestingly, in the zoomed-in view images ( Figure 3C), the MACA HaloTag ® -expression pattern on the PM of Myr (+) Ank GAG 1D4 expressing HeLa cells was characterised by dispersion all over the cell periphery as small, punctate dots. This pattern was totally different from that observed in HeLa cells expressing MACA-HaloTag ® alone or MACA-HaloTag ® and Ank A3 2D3, which showed smooth distribution throughout the PM ( Figure 3A,B, respectively). These observations showed that Ank GAG 1D4 disturbed the Gag distribution, suggesting the possibility that Ank GAG 1D4 disrupted the Gag polymerisation process. or Ank A3 2D3 (Gray bar) was added to H 6 MA-CA coated well and incubate for 1 h. The H 6 MA-CAbinding activity was detected by adding rabbit anti-ankyrin polyclonal antibody followed by goat anti-rabbit immunoglobulins-HRP. The data presented in the bar graph was formerly normalised with no ankyrin control. (B) The activity of HIV-1 protease was determined using ELISA. Plate was coated with the target of HIV-1 protease (recombinant H 6 MA-CA). Prior to adding HIV-1 protease, the coated plate was incubated with BSA (White bar), Ank GAG 1D4 (Black bar), or Ank A3 2D3 (nonbinding ankyrin protein) (Gray bar) for 1 h. The protease activity was determined by the presence of free C terminus of cleaved HIV-MA after cleaving with HIV-1 protease. The detection was measured by adding an anti-MA (HB-8975) monoclonal antibody (binds to the free C terminus of cleaved HIV-MA) followed by a goat anti-mouse immunoglobulins-HRP. The results represent as mean ± SD from a triplicate independent assay. *** p < 0.001; ns, not significant using one-way ANOVA.

Ank GAG 1D4 Affected RNA Packaging
HIV-1 Gag specifically recognises the FL RNA dimer in the cytosol and traffics to the PM as a Gag-FL RNA complex to join Gag-containing particles and displace RNAcontaining particles at assembly sites [4]. Additionally, other viral RNAs (MS, env) and cellular RNAs can be packaged into the virion. Since Ank GAG 1D4 modifies the Gag pattern at the PM, we tested whether Ank GAG 1D4 could also affect FL RNA packaging into new viral particles. To do so, HEK293T cells were co-transfected with pNL4-3∆env and pCEP4-Myr (+) Ankyrin (Ank GAG 1D4 or Ank A3 2D3)-GFP (0.5:1 molar ratio with pNL4-3∆env). After 24 h post-transfection, cells and virions were collected, then subjected to RNA extraction. RNA samples were studied by RT-qPCR analysis of viral (FL, MS, and env) and cellular (GAPDH, 7SL, and U6) RNAs. The result indicates that Ank GAG 1D4 and Ank A3 2D3 did not affect the levels of viral and cellular RNAs in transfected cells ( Figure 4A). The molar ratio of pNL4-3 ∆env to pCEP4-Myr (+) Ankyrin at 1:1 was not appropriate since the amount of generated virion was not sufficient for further analysis ( Figure 1A). we diminished the inhibitory effect of Ank GAG 1D4 in HIV-1 transfected cells, the molar ratio of pNL4-3∆env to pCEP4-Myr (+) Ankyrin was adjusted to 2:1 for sufficient virions collected to analyse their RNA contents by RT-qPCR. The results showed that both Ank GAG 1D4 and Ank A3 2D3 decreased the intravirion levels of viral RNAs (FL, MS, and env) and cellular RNAs (U6 small nuclear RNA (snRNA) and 7SL), compared to HEK293T cells expressing HIV-1 ∆env alone ( Figure 4B). Ank GAG 1D4 reduced the intravirion viral and cellular RNA levels much more than Ank A3 2D3 (except for 7SL RNA; Figure 4B). Additionally, viral release was measured by p24 ELISA. A control experiment was systematically performed without 10% Triton X-100 to control for the absence of cellular p24 contamination. As shown in Figure 4C, Ank GAG 1D4 decreased the production of viral progeny, even though it was not totally abolished because we adjusted the inhibitory effect of Ank GAG 1D4, as described above. Notably, without 10% Triton X-100, the p24 levels in all samples were undetectable, indicating that the p24 levels measured corresponded to intravirion p24. An equal number of viral particles (100 ng of p24) was subjected to compensate for the level of VLP-associated Gag. The level of differential RNA contents from the same number of virions was analysed. Both ankyrins negatively reduced the viral RNA contents ( Figure 4D) and affected the packaging of both viral RNAs and cellular RNAs ( Figure 4E). Interestingly, in comparison to Ank A3 2D3, Ank GAG 1D4 drastically decreased RNA packaging ( Figure 4E). Especially for the incorporation of FL HIV-1 RNA, which reflected the indispensable for infectious virion. However, Ank GAG 1D4 promoted significantly higher incorporation of 7SL cellular RNA into viral particles than did Ank A3 2D3. This result indicated that viral particles released from the Ank GAG 1D4-Gag producing cells lost their potential to encapsidate viral RNAs and cellular RNAs. concentrations in virion-containing supernatants were determined in the presence or absence of 10% TritonX-100, using a p24-ELISA. (D) The viral RNA contents were calculated by the percentage of total RNA level in virions at 100 ng of p24 (RNA copies/100 ng p24 × 100) normalised to HIV ∆env alone. (E) The packaging efficiency was calculated by the total viral RNA in virions relative to total cellular RNA and normalised to HIV ∆env alone. Data represent the mean ± SD from the triplicate independent assay. *** p < 0.001; ** p < 0.01; * p < 0.1; ns, not significant using one-way ANOVA.

Ank GAG 1D4 Restored CD81 Tetraspanin Localisation at the PM in Gag-Expressing Cells
During HIV-1 assembly and release, the tetraspanins transmembrane glycoprotein form dynamic networks with other proteins and HIV-1 proteins including HIV-1 Gag and Env at the PM [13,18,19]. Additionally, CD81 and CD9 tetraspanins expression on the PM were found to decrease during viral release [14] and were incorporated with released virions [18,22]. For this reason, we hypothesised that the distortion of Gag polymerisation by Ank GAG 1D4 could affect the CD81 tetraspanin remodelling at the PM. To address this issue, CD81 tetraspanins remodelling was assessed using conventional confocal microscopy. In this experiment, HeLa cells and ankyrin-expressing HeLa cells were transduced with VSV-G pseudotyped HIV-1 GagmCherry. At 48 h post-transduction, immunostaining was performed to demonstrate the distribution of CD81 tetraspanins under confocal microscopy. In this experiment, as Ank GAG 1D4 influenced the HIV-1 Gag distribution at the PM ( Figure 3C), tetraspanin recruitment to virus-assembly sites was considered. CD81 tetraspanin distribution in the PM of HeLa and ankyrin expressing HeLa cells was initially determined using confocal microscopy. Regarding confocal imaging, surface expression of CD81 remained with Myr (+) Ank GAG 1D4-EGFP or Myr (+) Ank A3 2D3-EGFP expressing HeLa cells ( Figure 5C,E, respectively).
Additionally, the level of overall surface CD81 expression in ankyrin-expressing cells was confirmed by flow cytometry. Accordingly, high expression levels of Ankyrin protein did not disturb the expression of CD81 at the PM ( Figure 6B). Therefore, the high-expressing Ankyrin protein was used in further study. Next, we analysed the surface expression of CD81 on HeLa cells expressing Gag-mCherry. Most of the HeLa transduced cells were positively expressed Gag-mCherry and were defined in 2 populations: cells with intermediate (mCherry+) and high (mCherry++) levels of Gag-mCherry expression ( Figure 6A). After staining with antihuman CD81, the surface CD81 expression disappeared on PM of HeLa cells expressing Gag-mCherry ( Figure 5B) and the CD81 intensity was decreased by 50% in cells strongly expressing Gag-mCherry as compared to the non-transduced cells ( Figure 6C). This result agreed with another study showing that CD9 and CD81 tetraspanin levels decreased in the PM at 48 h post-transfection in HeLa cells expressing Gag-GFP (14). Surprisingly, introducing HIV-1 Gag-mCherry into HeLa cells strongly expressing Ank GAG 1D4 cells efficiently maintained the CD81 tetraspanin on the PM up to 95% in the intermediate expression of Gag-mCherry and 86% in the high expression of Gag-mCherry ( Figure 6C). Nevertheless, the 80% (in the intermediate expression of Gag-mCherry) and 70% (in the high expression of Gag-mCherry) surface CD81 restoration were observed in HIV-1 Gag-mCherry transduced HeLa cells stably expressing Ank A3 2D3 ( Figure 6C). This phenomenon reflects the effect of the N-terminus myristoyl group of AnkA32D3 inserted into the inner leaflet of the cell membrane leading to nonspecifically disturbing of HIV-1 Gag assembly. Comparing the efficient restoration of surface CD81 between Ank GAG 1D4 and Ank A3 2D3 in HIV-1 Gag-mCherry transduced cells, the Ank GAG 1D4 was significantly sustained the CD81 distribution at the PM in both cells with intermediate (p < 0.001) and high (p < 0.001) levels of Gag-mCherry expression than Ank A3 2D3. These findings indicated that disturbing Gag distribution at PM by Ank GAG 1D4 impaired membrane remodelling during virus egress.  Relative surface CD81 intensity was calculated by the percentage of surface CD81 intensity of transduced cells and normalised against surface CD81 intensity of non-transduced cells. Data represent the mean ± SD from triplicate independent assay. Significant differences (*** p < 0.001; ns, not significant) were determined between transduced and non-transduced HeLa cells or ankyrinexpressing HeLa cells using one-way ANOVA. Ank GAG 1D4, Ank A3 2D3 represent HeLa cells stably expressing Myr (+) Ank GAG 1D4-EGFP and Myr (+) Ank A3 2D3-EGFP, respectively.

Discussion
Ank GAG 1D4 is a designed ankyrin repeat that binds a conserved sequence in the Nterminal region of the HIV-1 CA protein. Ank GAG 1D4 exhibits intracellular antiviral activity in the viral assembly process [37,38]. Recently, Ank GAG 1D4 exerts broad-spectrum antiviral activity by blocking the assembly of chimeric NL4-3-based virions derived from circulating strains among northern Thai patients [39]. Additionally, an affinity-improved Ank GAG 1D4 successfully demonstrated the enhancement of anti-HIV-1 activity in HIV-1 NL4-3 and HIV-1 maturation inhibitor-resistant (MIR) virus [42]. Although Ank GAG 1D4 performs a potent anti-HIV activity in HIV-1 infected cells, more understanding of molecular mechanisms which impaired HIV-1 production is required.
In this study, we showed that Ank GAG 1D4 did not interfere with the protease cleavage site, since the accumulation of the Pr55 Gag precursor and p41 proteins was observed inside the cells. Moreover, the CA p24 protein (the final product of Gag maturation) was detected in relatively few viral particles released from cells expressing Ank GAG 1D4. Moreover, this phenomenon was confirmed by directly detecting the free C-terminus epitope of cleaved HIV-MA generated by HIV-1 protease-cleaved H 6 MACA. This epitope was specifically captured by monoclonal antibody HB-8975 [43]. This data indicated that the interaction of Ank GAG 1D4 to HIV-1 CA did not hinder or alter the HIV-1 protease cleavage site on recombinant H 6 MACA.
The late stages of the HIV-1 life cycle include many events (such as intracellular trafficking, assembly, budding, release, and maturation) in which Gag plays a key role. Disturbing these processes diminishes the production of HIV-1 infectious particles. Inside Gag-producing cells, PI (4,5) P2 phospholipids interact with the HBR of MA and allosterically promote the release of sequestered myristate into lipid bilayers (2,3). The cytosolic, compact shape of Gag can be converted to an extended form and form polymers with other Gag proteins in the extended form [44]. Previously, using conventional pinhole confocal microscopy, the specific colocalisation of Myr (+) Ank GAG 1D4 and HIV-1 Gag at the PM of infected cells was observed [38]. Unexpectedly, Gag colocalisation with Myr (+) Ank A3 2D3, which does not bind to HIV-1 Gag, was also observed. Due to the diffraction limit of the light used for conventional confocal microscopy, it is not possible to distinguish spots less than 200 nm apart [45]. Thus, in this study, STED was used to overcome this limitation and monitor Gag distribution pattern at the PM. Previously, STED was employed to visualise the distribution of the Env glycoprotein on the surface of HIV-1 particles [46]. In addition, STED was recently used to demonstrate that HIV-1 Gag specifically restricts PI (4,5) P2 and cholesterol at assembly sites [47]. Using STED microscopy, we clearly observed that the MACA-HaloTag ® distributed continuously at the PM of MACA-HaloTag ® expressing cells. However, the continual pattern of MACA-HaloTag ® was clearly impaired in the presence of Ank GAG 1D4 ( Figure 3C). The apparent visualisation of dark gaps on the cell surface implied that embedding of Ank GAG 1D4 in the PM probably influenced the Gag polymerisation ( Figure 3C). To prove this, the electron microscopic (EM) analysis should be applied to assure the disturbance of MACA-HaloTag ® at the plasma membrane resulting in the disruption of CA lattice formation in defective viral progeny. Noticeably, the efficiency of HIV assembly was disrupted resulting in the reduction of viral progeny. Even though Ank GAG 1D4 efficiently reduces viral production, the effects of Ank GAG 1D4 on viral budding and membrane scission through the ESCRT machinery should be further investigated. ESCRT is not only associated with viral budding, but also participates in MVB biogenesis, which is important for transporting ubiquitinated protein for degradation [48]. As shown in Figure 3B, Myr + Ank A3 2D3 mainly localised in the cytoplasm. We inferred the involvement of the ESCRT degradation pathway, since Gag was not the specific target of Myr + Ank A3 2D3. Furthermore, the indirect disturbance of Gag-Gag multimersation by maturation inhibitor, bevirimat, caused abnormal virion morphology [49]. Likewise, the aberrant virion morphology caused by other CA, assembly, or maturation inhibitors has been well characterized and described by using electron microscopy [50][51][52]. Altering this critical step of HIV assembly by Ank GAG 1D4 possibly causes aberrant virion production. Investigation of the alteration of virion morphology produced by Ank GAG 1D4 expressing cells using EM should be further evaluated.
Because Ank GAG 1D4 did not significantly disturb the cellular FL HIV-1 RNA level, an effect on HIV-1 RNA transcription was excluded. Therefore, accumulation of the Gag polyprotein inside Ank GAG 1D4 expressing cells resulted from the retardation of the Gag polyprotein-release rate during the assembly process. As the extended Gag conformation generally uses the NC domain to recruit FL RNA and initiate the viral-assembly stage [53]. RNA levels in virions and RNA-packaging efficiency have been studied by RT-qPCR (reviewed in [54]). In this study, Gag clearly lost its ability to specifically incorporate FL HIV-1 RNA into virions in the presence of Ank GAG 1D4 ( Figure 4D,E). The ability of Gag to multimerize on RNA via the NC domain is important for RNA packaging. Since NC is not the target of Ank GAG 1D4, the mechanism of Ank GAG 1D4 should rather participate in the disturbance of Gag multimerisation. The networking of Gag is crucial for successfully stabilizing RNA at the plasma membrane in the preassembly stage [53,55]. According to STED analysis, the impairment of Gag distribution in Ank GAG 1D4 expressing cells was indicated, thus, reflecting the interference of the Gag polymerisation process. In general, FL HIV-1 RNA and spliced RNA can be packaged into virions due to the presence of an internal loop and the lower part of SL1 in the 5 -untranslated region of HIV-1 RNA. However, FL HIV-1 RNA is preferably selected by the Gag precursor with higher affinity than spliced RNA due to the counterbalance-regulation domain spanning nucleotides downstream of SL4 and upstream of SL1, and the specific region between nucleotides 355-400, which is not present in spliced RNA [8,10]. Accordingly, the presence of Ank GAG 1D4 also interfered with the incorporation of spliced RNAs (MS and env RNA) and cellular RNAs (U6 snRNA and 7SL) into nascent virions. Data from several studies showed that HIV-1 particles can specifically package small Pol-III transcripts in addition to FL RNA [11]. Ank GAG 1D4-Gag expressing cells not only lost HIV-1 RNA encapsidation, but also cellular RNA incorporation ( Figure 4D,E). Compared to Ank A3 2D3, the FL RNA reduction induced by Ank GAG 1D4 was accompanied by increased 7SL RNA incorporation into virions ( Figure 4D,E). Interestingly, these opposing effects on the FL and 7SL RNA packaging levels were also reported when the packaging signal was deleted from FL RNA [9]. Additionally, increasing 7SL RNA incorporation in virions was possibly caused by Gag accumulation induced by Ank GAG 1D4, since Gag accumulation inside producer cells previously promoted 7SL RNA incorporation into virions [56]. A partial reduction of the RNA-packaging efficiency was also observed in the presence of Ank A3 2D3, which may have been caused by competition between the myristoyl group of Ank A3 2D3 and Gag in recruiting cholesterol molecules [57]. In contrast to Ank A3 2D3, a tremendously impaired RNA packaging in Ank GAG 1D4 expressing cells was significantly observed. Additionally, it was clear that the alignment of Gag at PM in Ank A3 2D3-Gag-expressing cells was not disturbed ( Figure 3B), as Gag is not the specific target of Ank A3 2D3. Thus, RNA incorporation partially occurred in the presence of Ank A3 2D3.
The clustering of tetraspanins with Gag and Env at the PM has been observed in diverse types of HIV-1 infected cells [18,58]. Gag polymerisation at the PM can induce the formation of microdomains, including TEMs [59]. TEMs have been proposed as gateways for HIV-1 assembly and budding [14,60]. We observed that at 48 h post-transduction with VSV-G-pseudotyped HIV-1 GagmCherry, the CD81 tetraspanin vanished from the PM. Diminishing PM expression of the CD81 tetraspanin in HIV-1-infected cells was related to VLP release [14]. Interestingly, we also observed that inhibiting HIV-1 egress with Ank GAG 1D4 caused a rebound of CD81 tetraspanin on the PM of HIV-1 Gag-expressing cells. These data indicated that Ank GAG 1D4 caused membrane remodelling during viral release, since Gag polymerisation was disturbed. Beyond Ank GAG 1D4, no other small molecules or peptide inhibitors targeting CA have been reported to affect the remodelling of tetraspanins on the PM. Moreover, the presence of tetraspanins at exit sites can reduce syncytial formation in virus-producing cells and cell-to-cell fusion induced by the virus [61,62]. Because Ank GAG 1D4 is capable of preventing syncytial formation [37][38][39], it is worth further investigating its utility in blocking viral transfer via cell-to-cell contact.

Conclusions
The data obtained in this study illustrate the molecular mechanisms whereby Ank GAG 1D4 blocks HIV replication. The intracellular expression of Ank GAG 1D4 disrupted the primary checkpoint at the late stages of viral progeny production when the Gag polyprotein formed. The resulting intracellular accumulation of the Gag polyprotein was followed by inefficient Gag assembly and decreased RNA packaging. The disruption of CD81 tetraspanin membrane remodelling is proposed as a significant marker of defective HIV replication. Taken together, these findings could influence future strategies for using the altered scaffold protein as an anti-HIV-1 molecule.